Application Note
0704
Fatty Acids Analysis Using LDTD-APCI MS/MS in Negative and Positive Chemical Ionization Pierre Picard, Patrice Tremblay, Sylvain Letarte
Keywords: LDTD-APCI, Oleic Acid, Stearic Acid
Phytronix Technologies, Québec City, CANADA
Introduction
LDTD ionization process
Fatty Acids analysis is a great concern as being present in food products, plant oil and in living organism. They are also precursors in the synthesis of triacylglycol and cholesterol. A large variety of analytical methods are available for the characterization, quantification and detection of Fatty Acids in biological matrixes1-3. GC-MS analysis for Fatty Acids require long-sample preparation including liquid-liquid extraction and chemical derivatization steps. The use of HPLC-MS for Fatty Acids analysis is generally associated to inefficient LC separation and/or time-consuming separation time.
The LDTD source uses an infrared laser diode to desorb samples, previously dried onto a well of the LazWell™ (96-well plate). The desorbed gas phase molecules are carried over by a carrier gas into a corona discharge region to undergo APCI and then transferred directly into the mass spectrometer. Samples Preparation Oleic acid and Stearic acid were respectively dissolved in ethanol and chloroform at different concentrations. Samples were directly deposited into the LazWellTM plate without any sample preparation such as chemical derivatization.
Fatty Acids are analyzed using the LDTD-APCI source without chemical derivatization and chromatographic separation in positive and negative chemical ionization mode. The MS/MS detection of Oleic and Stearic acids will also be presented.
LDTD Parameters Stabilization Time Laser Power Pattern
Goals •
Illustrate the efficiency of the LDTD-APCI source for Fatty Acids screening,
•
Demonstrate the versatility of the LDTD-APCI source for negative and positive Fatty Acids chemical ionization.
Carrier Gas Flow Carrier Gas Temperature MS Parameters Collision Gas Pressure Scan Time Q1 Width Q3 Width Needle Voltage Capillary Temperature Sweep Gas Flow
Instrumentation •
Phytronix Technologies LDTD ionization source (model T-960),
•
Thermo Fisher Scientific TSQ QuantumTM Ultra AM mass spectrometer. Oleic acid 99 % and Stearic acid 99 % were purchased from Alfa Aesar, MA, USA.
•
All other chemicals or reagent are HPLC grade.
Ionization mode Stearic acid Oleic acid
Fatty Acids
1.2 mTorr Argon 0.1 sec 0.3 amu 0.7 amu 4500 V 250 oC 1 arbitrary unit
Scan Parameters
Chemicals •
3 sec 0 to 40 % in 2 sec Hold at 40 % for 3 sec 2.0 mL/min 50 oC
1 of 4
+ + -
Precursor Ion (m/z) 285 283 283 282
Product Collision Ion 1 Energy (V) 57 20 265 35 97 22 263 30
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The LDTD-APCI source is used to analyse Stearic acid in both positive and negative ionization mode. As shown in Fig. 1, the major ions are the pseudomolecular ions [M+H]+ and [M-H]-. The LDTD-APCI source allows to analyse Stearic acid in positive chemical ionization (Fig.1B) which is not reported in recent litterature. Moreover, the spectra confirm that no thermal byproduct was produced during the thermal desorption process. This is a powerfull advantage of the LDTD-APCI source to performe Fatty acids analysis without any chemical derivatization.
Stearic Acid Analysis Stearic acid M.W. 284.47 g/mol o Boiling Point : 383 C [M-H]-
stearic_prod_NEG #19 RT: 0.16 AV: 1 NL: 2.70E8 T: - p ESI Q1MS [30.000-400.000] 100
285.19
100
A
90
90
85
85
80
80
75
75
70
70
65
65
60
60
55 284.28 50 45
285.05
B
95
Relative Abundance
Relative Abundance
95
[M+H]+
stearic_full_POS #46 RT: 0.37 AV: 1 NL: 1.36E8 T: + p ESI Q1MS [30.000-400.000]
283.02
55
[M-H2O+H]+
50
267.06
45
40
40
35
35
30
30
25
25
20
20
286.10
286.52
15
15
314.31 10
10
281.20
5 0
60.10 50
100
150
200
250
300
350
249.21
5
315.29 339.15 383.39
253.41 98.87 126.94 169.21 197.14 239.21
71.02 96.91
111.12
287.50
122.95
200.99 239.07
295.13
0
400
50
100
150
200
m/z
250
341.25 356.02
300
350
400
m/z
Figure 1 Full scan MS spectra obtained for LDTD-APCI analysis of Stearic acid in A) positive APCI, and in B) negative APCI [M-H]-
stearic_prod_NEG3 #25-34 RT: 0.19-0.26 AV: 10 SM: 7G NL: 4.78E5 T: - p ESI Full ms2
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283.47
100
A
283.17
90
90
85
85
80
80
75
75
70
70
65
65
60
60
55
[M-H2O-H]-
50
265.67
45
B
95
Relative Abundance
Relative Abundance
95
stearic_prod_POS #23 RT: 0.17 AV: 1 NL: 1.07E8 T: + p ESI Full ms2
[email protected] [30.000-300.000] 57.20 100 57.40
40
71.19
[M+H]+
103.09
285.17
103.39
55
The low background associated to the LDTD-APCI source allows Fatty Acids analysis in MS/MS. The obtained spectra (Fig. 2) show low fragmentation when Stearic acid is analyzed under negative APCI as reported in the litterature. However, under positive APCI conditions, the fragmentation pattern (Fig. 2B) in combinaison to the high intensity signal allows using MS/MS for Stearic acid identification and quantification. Moreover, it enhences the selectivity of the method.
284.97
50 45 40
265.07 35
35
30
30
25
25
43.20
117.39
20
20
263.57
15
15
10
10
80.09 5
45.40
69.09
83.49
127.19 140.59
210.98 225.27 245.57 169.58 182.88
50
100
150
200
250
149.28 149.48 163.28
201.28
215.48 247.37
5
291.76
0
135.59
300
265.57 286.37
0 50
100
150
200
250
300
m/z
m/z
Figure 2 MS/MS spectra for Stearic analysis with the LDTD-APCI source in A) negative APCI and, in B) positive APCI
For more information about your specific application, visit www.phytronix.com/science Fatty Acids
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Oleic Acid Analysis [M-H]-
oleicFULLneg #22-25 RT: 0.18-0.20 AV: 4 SM: 7G NL: 5.95E6 T: - p ESI Q1MS [30.000-400.000]
95
A
95 90
85
85
80
80
75
+
[M-H2O+H]
B
284.14
75 255.30
70 65
70
Relative Abundance
60
55 50 45
55 50 45
40
40
35
35
30
30
25
25
20
20
15
247.11
15
284.28 227.17
10
297.16
10
199.10
0
Oleic acid is analyzed in both positive and negative ionization mode with the LDTD-APCI source. As shown in Fig. 3, the major ions are the pseudomolecular ions [M+H]+ and [M-H]-. As for Stearic acid, the LDTDAPCI source allows to analyse Oleic acid in positive chemical ionization (Fig. 3B).
65
283.37
60 Relative Abundance
265.10 283.09
100
90
5
[M+H]+
oleicFULLpos7 #28 RT: 0.22 AV: 1 SM: 7G NL: 2.61E8 T: + p ESI Q1MS [30.000-400.000]
281.34
100
Oleic acid M.W. 283.2559 g/mol o Boiling Point : 360 C
60.03 46.10 50
92.99
295.27 311.37
141.22 171.03
5 363.16
61.01 57.09
110.98 153.12
185.24
100
150
200
250
300
350
50
100
150
The low background associated to the LDTD-APCI source allows Oleic acid analysis in MS/MS. The obtained spectrum when Oleic acid was analyzed under negative APCI conditions shows low fragments intensity (Fig. 4A). This observation explains why MS/MS analysis is not reported on underivatized Oleic acid. However, with positive APCI, the fragmentation pattern (Fig. 4B) offers interesting MS/MS transitions that can be used for Oleic acid analysis. Moreover, it enhances the selectivity of the method.
199.17 213.10 298.07
341.18 359.17
0 200
m/z
250
300
350
400
m/z
Figure 3 Full scan MS spectra obtained for LDTD-APCI analysis of Oleic acid in A) positive APCI, and in B) negative APCI [M-H]-
oleic prod neg_070216120745 #57-61 RT: 0.47-0.50 AV: 5 SM: 7G NL: 1.07E6 T: - p ESI sid=5.00 Full ms2
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281.13
100
A
95
90
90
85
85
80
80
75
75
70
70
65
65
60
60 Relative Abundance
Relative Abundance
95
oleicPRODpos7 #20-26 RT: 0.16-0.21 AV: 7 NL: 5.38E7 T: + p ESI Full ms2
[email protected] [30.000-300.000] 97.12 100
55 50 45
55
97.40
B
121.13 135.20
69.13 69.34
149.27 57.23 57.09
149.41
50 45
40
40
35
35
30
30
25
25
20
20
15
15
10
10
55.27
163.34
177.19
0
59.47 50
71.23
96.77 100
127.29 150
200
250
m/z
[M-2H2O+H]+
177.47
247.54
191.12
-
[M-H2O+H]+
205.33
[M-H O-H]
2 182.30 208.90 263.21 167.88 183.35 226.12
5
43.23
5 282.11 300
265.17
223.39
[M+H]+
282.88
0 50
100
150
200
250
300
m/z
Figure 4 MS/MS spectra for Oleic acid analysis with the LDTD-APCI source in A) negative APCI and, in B) positive APCI
For more information about your specific application, visit www.phytronix.com/science Fatty Acids
337 rue Saint-Joseph E. Québec, Qc Canada G1K 3B3 Phone : +1 (418) 692-1414 Fax : +1 (418) 692-4940
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Table 1 Fatty Acids LOD and LOQ obtained with the LDTD-APCI source
Quantitative analysis of Stearic and Oleic acid The fragmentation obtained with positive APCI conditions for both Fatty Acids analysis (Figs 1B, and 3B) associated with high signal to noise ratio are the reasons why Fatty Acids are quantified in positive APCI using the LDTD-APCI source. The MS/MS transitions were optimized to obtain a good signal-to-noise ratio and allow an excellent linearity over the studied concentration range (Figs. 5, and 6). The LOD and LOQ achieved are reported in Table 1 and are in the concentration range found in current literature. Good repeatability was also obtained for all concentrations. Positive APCI MS/MS analysis on Fatty Acids has not been reported in the literature and the presented results enhance the selectivity of Fatty Acids analysis.
Stearic acid Oleic acid
MS/MS Transition : 285 → 57 amu
35000000
Area (count)
8
27
2.7
9
Fatty Acids analysis using HPLC-MS are timeconsuming and offer a low level of selectivity. LDTDAPCI technology allows to easily identifying Fatty Acids in both negative and positive APCI without any timeconsuming chemical derivatization. Due to the low number of fragments produced with negative APCI, positive APCI was selected for Fatty Acids quantification. Good linearity was obtained and allows low LOD and LOQ values for Stearic and Oleic acid. The thermal desorption process produces clean Fatty Acids MS spectra.
2
40000000
LOQ (ng/mL)
Conclusions
r = 0.999
45000000
LOD (ng/mL)
30000000 25000000 20000000 15000000 10000000
References
5000000
1
0 0
20
40
60
80
100
Stearic acid concentration (µg/mL)
Gagné et al., 2007, J. Lipid Res., 48, 252. Lin et al., 2005, J. Lipid Res., 46, 1974. 3 Nagy et al., 2004, Anal. Chem., 76, 1935. 2
Figure 5 Calibration curve for Stearic acid analysis using the LDTD-APCI source 40000000
MS/MS Transition : 283 → 97 amu
35000000
2
r = 0.999
Area (count)
30000000 25000000 20000000 15000000 10000000 5000000 0 0
2
4 6 8 10 Oleic acid concentration (µg/mL) Figure 6 Calibration curve for Oleic acid analysis using the LDTD-APCI source
For more information about your specific application, visit www.phytronix.com/science Fatty Acids
337 rue Saint-Joseph E. Québec, Qc Canada G1K 3B3 Phone : +1 (418) 692-1414 Fax : +1 (418) 692-4940
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